The ability of duplex RNA to recognize mRNA and silence gene expression through post-transcriptional RNA interference (RNAi) is widely appreciated (Tang, 2004). Short interfering RNAs (siRNAs) have become common laboratory tools for controlling gene expression and endogenously expressed microRNAs (miRNAs) participate in an expanding array of cellular pathways.
RNA-directed DNA methylation was described originally in plants (Matzke et al 2004), where it was found that RNA viruses and viroids could induce methylation in genomic DNA sequences (Massenegger et al 1994). Methylated bases were concentrated within sequences of DNA that were complementary to RNA, suggesting a sequence-specific mechanism for recognition (Pelissier and Wassenegger 2000).
In yeast, small RNAs that target centromere repeat sequences and mating type loci can silence gene expression by promoting modification of heterochromatin (Grewal and Moazed 2003; Bernstein and Allis 2005). Chromatin modifications involve methylation of histone H3 at Lysine 9 (Volpe et al 2002) and require RNA-dependent RNA polymerase (Sugiyama et al 2005) and DNA polymerase II (Schramke et al 2005). Modification involves proteins of the RNA-induced transcriptional silencing (RITS) pathway (Verdel et al 2003) including argonaute 1 (Sigova et al 2004), a member of a protein family that is also involved in post-transcriptional silencing.
Recently, several reports have suggested that antigene RNAs (agRNAs)—short oligonucleotides that target chromosomal DNA—can also silence expression in mammalian cells. Kawasaki and Taira targeted ten duplex RNAs to sequences within the E-cadherin promoter that contained CpG dinucleotides (Kawasaki and Taira, 2004). DNA methylation was observed at all of these sites. Individual RNAs yielded only marginal reductions in E-cadherin expression but more complete silencing could be achieved if all ten RNAs were combined. A link between methylation and silencing was supported by the observation that duplex RNAs were not able to inhibit expression of E-cadherin when methyl-transferase genes DMNT1 and DMNT3B were silenced.
In a similar study, Morris and co-workers demonstrated that duplex RNAs targeting the promoter of Elongation factor 1α (EF1A) could inhibit expression (Morris et al, 2004). They observed methylation of DNA at the target sequence and that addition of the methylation inhibitor 5′-aza-2′-deoxycytidine (5-aza-dC) in conjunction with the histone deacetylase inhibitor trichostatin (TSA) reversed silencing. The studies from the Taira and Morris laboratories were significant because they provided evidence that RNA could target DNA for silencing in mammalian cells and suggested that RNA could induce DNA methylation. In the Morris study silencing by a synthetic agRNA required use of a peptide designed to promote nuclear uptake, but other studies have suggested that standard transfection procedures are adequate (Kawasaki and Taira 2004; Castanotto et al 2005; Janowski et al 2005; Ting et al 2005).
Other attempts to achieve RNA-directed methylation in mammalian cells have been less successful. Steer and coworkers tested RNAs that targeted the gene encoding Huntingtin and did not detect any methylation (Park et al 2004). No RNA-directed methylation was observed upon stable expression of double-stranded RNA in mouse oocytes (Svoboda, P. et al 2004). Rossi and colleagues used expressed short hairpin RNAs (shRNAs) to target a well-characterized CpG island within the promoter for the tumor suppressor RASSF1A (Castanotto et al 2005). They reported modest inhibition of gene expression. The methylation-specific PCR assay showed methylation, but the more complete bisulphite sequencing assay did not.
Our laboratory discovered that efficient RNA-mediated silencing of chromosomal DNA can be achieved independent of DNA methylation (Janowski et al 2005; U.S. Pat. appl No. 60/661,769). We targeted transcription start sites to block expression by obstructing the initiation of transcription. A practical advantage of targeting transcription start sites is that they occur in all genes and provide a general and predictable class of target sequences; targeting transcription start sites would also be expected to block gene expression regardless of whether methylation occurs.
In contrast to other studies we observed no methylation by methylation-specific PCR or sodium bisulfite sequencing. Inhibition of methyl transferase activity using 5-azaC or an anti-methyl transferase siRNA had no effect on gene silencing, suggesting that methylation was not involved in silencing. The silencing we observed was more potent than that reported in prior studies, indicating that transcription start sites may be particularly susceptible targets for agRNAs.
Baylin and colleagues revisited transcriptional silencing of E-cadherin (Ting et al 2005). They observed efficient silencing of gene expression when two promoter-targeted duplex RNAs were used in tandem, but not when the RNAs were used individually. Baylin observed no evidence for DNA methylation.
It has been reported that siRNAs targeting the E-cadherin gene promoter can activate transcription (Li et al, 2005) in cultured breast cancer cells. Similarly, data has been presented indicating increased EF1A mRNA expression by promoter-targeted siRNA (Morris et al 2004; see FIG. 3A, first two bars). It has also been reported that nuclear localized small modulatory double-stranded (ds) RNA (smRNA) coding NRSE sequences triggered activation of transcription of NRSE genes in adult hippocampal neural stem cells (Kuwabara et al, 2004; and Kuwabara et al, 2005).